Array systems and methods

ABSTRACT

Methods, kits, arrays, and biosensors for detecting proteins, modified-proteins, protein-protein interactions, protein-DNA interactions, autoantibodies, and protein-small molecule interactions, are disclosed. A representative method of detecting proteins of the present invention includes exposing a solid support to a solution containing proteins; conjugating proteins to the solid support; exposing the solide support to a plurality of types of DNA-conjugated antibodies, wherein each type of DNA-conjugated antibody has an affinity for a specified protein; forming a complex between a protein conjugated with the solid support and a type of DNA-conjugated antibody when the protein is the specified protein for which the DNA-conjugated antibody has an affinity; separating the complex from the solution of proteins and the DNA-conjugated antibodies; releasing DNA from the DNA-conjugated antibodies; and detecting the DNA, wherein each DNA indicates the presence of the specified proteins.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to copending U.S. provisionalapplication entitled, “Immuno-DNA Array Systems and Methods,” havingSer. No. 60/338,601, filed Nov. 13, 2001, which is entirely incorporatedherein by reference.

TECHNICAL FIELD

The present invention is generally related to the analysis of proteinsand polypeptides and, more particularly, is related to systems andmethods for the simultaneous detection of detecting proteins and proteininteractions.

BACKGROUND OF THE INVENTION

All cell functions, including cell proliferation, cell death, and alldifferentiation, as well as maintenance of health status and developmentof disease are controlled by many genes and signaling pathways. Newtechniques such as cDNA microarrays have enabled the analysis of theglobal gene expression. DNA microarray technology permits systematicapproaches to biological discovery that has a profound impact on cancerresearch. The ability to obtain global gene expression profiles promisesto be an exceptionally powerful means to explore basic biology,facilitate drug discovery, provide new diagnostic tools for diseases,and tailor therapeutics to specific gene profiles. For instance, inbasic cancer biology, the global analysis of gene profiles in cancercells can uncover crucial clues to the underlying changes in geneticnetworks and programs of malignantly transformed cells. In addition, DNAmicroarrays are useful for classifying human diseases. These types ofgene profiles provide valuable information about the molecularmechanisms responsible for disease development, disease diagnosis, andpatient prognosis.

Although DNA microarray analysis of global gene expressions holds greatpromise in the fight against human disease, proteins do almost all ofthe work in the cell.

Experimental evidence clearly shows a disparity between the relativeexpression levels of mRNA and their corresponding proteins. Moreimportantly, protein-protein interactions, protein modification, andprotein-DNA interactions are important concepts for studying howproteins perform their functions, which cannot be studied by DNA alone.

Protein analysis is thereof important to understanding these concepts.In some cases such as cytokines and growth factors, protein analysis iseasier to perform than genomic analysis. Further, protein analysis maybe the only effective way to analyze the specific antibody levels.Therefore, to effectively treat cancer, a complete picture of theprotein profile is desired. Unfortunately, unlike the cDNA microarraytechnology, the methodology that allows detecting an entire pool ofproteins does not exist.

Currently, two-dimensional polyacrylamide gel electrophoresis (twodimensional gel system) coupled with mass spectrometry is the mainstreamapproach to analyze multiple protein expressions. However, this approachsuffers from several problems such as requiring sophisticated devices,having low sensitivity, and having a lack of a qualification process ofthe protein. Some proteins cannot be identified using this approach. Forexample, low molecular weight proteins are difficult to quantify. Inaddition, the detection limit of the two dimensional gel system is atthe nanogram level.

Unfortunately, many important proteins express much lower levels thanthe two-dimensional gel system can detect. Therefore, a heretoforeunaddressed need exists in the industry to develop a new approach toassess proteins.

SUMMARY OF THE INVENTION

Embodiments of the present invention include methods, kits, arrays, andbiosensors for detecting proteins, modified-proteins, protein-proteininteractions, protein-DNA interactions, autoantibodies, andprotein-small molecule interactions. A representative method ofdetecting proteins of the present invention includes exposing a solidsupport to a solution containing proteins; conjugating proteins to thesolid support; exposing the solid support to a plurality of types ofDNA-conjugated antibodies, wherein each type of DNA-conjugated antibodyhas an affinity for a specified protein; forming a complex between aprotein conjugated with the solid support and a type of DNA-conjugatedantibody when the protein is the specified protein for which theDNA-conjugated antibody has an affinity; separating the complex from thesolution of proteins and the DNA-conjugated antibodies; releasing DNAfrom the DNA-conjugated antibodies; and detecting the DNA, wherein eachDNA indicates the presence of the specified proteins.

A representative method of detecting protein-protein interactionsincludes exposing a solid support to a plurality first proteins, whereinthe plurality of first proteins conjugate with the solid support;exposing the solid support to a second solution of proteins, wherein theproteins in the second solution are capable of conjugating with theplurality of first proteins; exposing the solid support to a pluralityof types of DNA-conjugated antibodies, wherein each type ofDNA-conjugated antibody has an affinity for a specified protein; forminga complex between a protein in the second solution that has conjugatedwith the first protein and a type of DNA-conjugated antibody when theprotein is the specified protein for which the DNA-conjugated antibodyhas an affinity; separating the complex from the solution of firstproteins, second proteins, and the DNA-conjugated antibodies; releasingDNA from the DNA-conjugated antibodies; and detecting the DNA, whereineach DNA indicates the presence of the specified proteins whichindicates that the specified proteins interacted with the first protein.

A representative method of detecting protein-DNA interactions includesexposing a solid support to a first DNA having at least one portion,wherein the first DNA conjugates with the solid support; exposing thesolid support to a solution of proteins, wherein the proteins in thesolution are capable of conjugating with a portion of the first DNA;exposing the solid support to a plurality of types of DNA-conjugatedantibodies, wherein each type of DNA-conjugated antibody has an affinityfor a specified protein; forming a complex between a protein in thesolution that has conjugated with a portion of the first DNA and a typeof DNA-conjugated antibody when the protein is the specified protein forwhich the DNA-conjugated antibody has an affinity; separating thecomplex from the solution of proteins and the DNA-conjugated antibodies;releasing DNA from the DNA-conjugated antibodies; and detecting the DNA,wherein each DNA indicates the presence of the specified proteins, whichindicates that the specified proteins interacted with the portion ofDNA.

A representative method of detecting modified proteins includes exposinga solid support to a solution containing modified-proteins; conjugatingmodified-proteins to the solid support; exposing the solid support to aplurality of types of DNA-conjugated antibodies, wherein each type ofDNA-conjugated antibody has an affinity for a specifiedmodified-protein; forming a complex between a modified-proteinconjugated to the solid support and a type of DNA-conjugated antibodywhen the modified-protein is the specified modified-protein for whichthe DNA-conjugated antibody has an affinity; separating the complex fromthe solution of modified-proteins and the DNA-conjugated antibodies;releasing DNA from the DNA-conjugated antibodies; and detecting the DNA,wherein each DNA indicates the presence of the specifiedmodified-proteins.

A representative method of detecting autoantibodies includes exposing asolution containing proteins to a solution containing a plurality ofautoantibodies, wherein each autoantibody has an affinity of a specifiedprotein; forming a first complex between a protein in the solution and atype of autoantibody when the protein is the specified protein for whichthe autoantibody has an affinity; exposing the first complex to asolution containing a plurality of types of DNA-conjugated antibodies,wherein each type of DNA-conjugated antibody has an affinity for aspecified protein; forming a second complex between first complex and atype of DNA-conjugated antibody when the protein is the specifiedprotein for which the DNA-conjugated antibody has an affinity; exposingthe second complex to a solution having a plurality of antibodies,wherein each type of antibody has an affinity for a specifiedautoantibody; forming a third complex between the second complex and atype of antibody when the autoantibody is the specified antibody forwhich the antibody has an affinity; separating the third complex fromthe solutions; releasing DNA from the DNA-conjugated antibodies; anddetecting the DNA, wherein each DNA indicates the presence of specifiedautoantibodies.

A representative method of detecting protein-small molecule interactionincludes exposing a support to a plurality small molecules, wherein theplurality of small molecules conjugate with the support; exposing thesupport to a second solution of proteins, wherein each of the proteinsin the second solution are capable of conjugating with a specified smallmolecule; forming a first complex between the small molecule and a typeof protein, when the small molecule is the specified small molecule forwhich the protein has an affinity; exposing the support to a pluralityof types of DNA-conjugated antibodies, wherein each type ofDNA-conjugated antibody has an affinity for a specified protein; forminga second complex between a protein conjugated to the support and a typeof DNA-conjugated antibody, when the protein is the specified proteinfor which the antibody has an affinity; separating the second complexfrom the solution of proteins and the DNA-conjugated antibodies;removing DNA from the DNA-conjugated antibodies; and detecting the DNA,wherein each DNA indicates the presence of the specified proteins, whichindicates that the specified proteins interacted with the smallmolecules.

Other systems, methods, features, and advantages of the presentinvention will be or will become apparent to one with skill in the artupon examination of the following drawings and detailed description. Itis intended that all such additional systems, methods, features, andadvantages be included within this description, be within the scope ofthe present invention, and be protected by the accompanying claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Many aspects of the invention can be better understood with reference tothe following drawings. The components in the drawings are notnecessarily to scale, emphasis instead being placed upon clearlyillustrating the principles of the present invention. Moreover, in thedrawings, like reference numerals designate corresponding partsthroughout the several views.

FIGS. 1A and 1B are schematic diagrams that illustrate a representativeembodiment of an immuno-DNA assay system for detecting proteins.

FIGS. 2A and 2B are schematic diagrams that illustrate anotherrepresentative embodiment of the immuno-DNA assay system for detectingprotein modifications.

FIGS. 3A-3C are schematic diagrams that illustrate a representativeembodiment of the immuno-DNA assay system for assessing protein-proteininteractions.

FIGS. 4A-4C are schematic diagrams that illustrate a representativeembodiment of the immuno-DNA assay system for assessing DNA-proteininteractions.

FIGS. 5A and 5B are schematic diagrams that illustrate a representativeembodiment of the immuno-DNA assay system for assessing proteins thatinhibit protein-protein interactions.

FIGS. 6A-6C are schematic diagrams that illustrate a representativeembodiment of the immuno-DNA assay system for assessing proteins thatinhibit DNA-protein interactions.

FIGS. 7A-7C are schematic diagrams that illustrate a representativeembodiment of the immuno-DNA assay system for assessingantibody-autoantibody interactions.

DETAILED DESCRIPTION

Definition of Terms

“DNA” (deoxyribonucleic acid) generally refers to any polynucleotide.“DNA” includes, without limitation, single- and double-stranded DNA; DNAthat is a mixture of single- and double-stranded regions; single- anddouble-stranded ribonucleic acid (RNA); RNA that is mixture of single-and double-stranded regions; and hybrid molecules comprising DNA and RNAthat may be single-stranded or, more typically, double-stranded or amixture of single- and double-stranded regions. In addition, “DNA”refers to triple-stranded regions comprising RNA or DNA, or both RNA andDNA. The term “DNA” also includes DNAs or RNAs containing one or moremodified bases and DNAs or RNAs with backbones modified for stability orfor other reasons. “Modified” bases include, for example, tritylatedbases and unusual bases such as inosine. A variety of modifications maybe made to DNA and RNA; thus, “DNA” embraces chemically, enzymatically,or metabolically modified forms of polynucleotides as typically found innature, as well as the chemical forms of DNA and RNA characteristic ofviruses and cells. “DNA” also embraces relatively short polynucleotides,often referred to as oligonucleotides.

“Protein” refers to any peptide, polypeptide, or protein comprising twoor more amino acids joined to each other by peptide bonds or modifiedpeptide bonds, (i.e., peptide isosteres). “Protein” refers to both shortchains (commonly referred to as peptides, oligopeptides, or oligomers)and to longer chains generally referred to as proteins. “Protein” maycontain amino acids other than the 20 gene-encoded amino acids.“Protein” includes amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques, which are well known in the art. Suchmodifications are described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.

The term “antibody” includes reference to antigen binding forms ofantibodies (e.g., Fab, F(ab)₂). The term “antibody” frequently refers toa polypeptide substantially encoded by an immunoglobulin gene orimmunoglobulin genes, or fragments thereof, which specifically bind andrecognize an analyte (antigen). However, while various antibodyfragments can be defined in terms of the digestion of an intactantibody, one of ordinary skill in the art will appreciate that suchfragments may be synthesized de novo either chemically or by utilizingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments such as single chain Fv, chimericantibodies (i.e., comprising constant and variable regions fromdifferent species), humanized antibodies (i.e., comprising acomplementarity determining region (CDR) from a non-human source) andheteroconjugate antibodies (e.g., bispecific antibodies). In particular,autoantibodies are antibodies that react with a constituent of thetissue of the animal.

The term “antigen” includes reference to a substance to which anantibody can be generated and/or to which the antibody is specificallyimmunoreactive. The specific immunoreactive sites within the antigen areknown as epitopes or antigenic determinants. These epitopes can be alinear array of monomers in a polymeric composition-such as amino acidsin a protein-or consist of or comprise a more complex secondary ortertiary structure. Those of ordinary skill in the art will recognizethat all immunogens (i.e., substances capable of eliciting an immuneresponse) are antigens; however some antigens, such as haptens, are notimmunogens, but may be made immunogenic by being coupled to a carriermolecule. An antibody immunologically reactive with a particular antigencan be generated in vivo or by recombinant methods such as selection oflibraries of recombinant antibodies in phage or similar vectors. See,e.g., Huse et al., Science 246: 1275-1281 (1989); and Ward, et al.,Nature 341: 544-546 (1989); and Vaughan et al., Nature Biotech. 14:309-314 (1996).

Discussion

The present invention provides immuno-DNA array systems and methods forthe analysis of polypeptides and proteins (hereinafter “proteins”).Specifically, embodiments of the present invention include methods,kits, assays, and biosensors for detecting proteins, modified-proteins,protein-protein interactions, protein-DNA interactions, autoantibodies,and protein-small molecule interactions. For example, embodiments of theimmuno-DNA array system are capable of determining the presence of aprotein and/or protein modification and are also capable of assessingprotein-protein interactions, DNA-protein interactions,inhibitor-protein interactions, inhibitor-DNA interactions, andautoantibodies.

Generally, the immuno-DNA array system involves immobilization of one ormore specified proteins onto one or more solid supports. The specifiedproteins bound to the solid support are then contacted with one or moreDNA-conjugated antibody types. Each of the DNA-conjugated antibody typesconjugate (e.g., bond, bind, chemically attached or associated with)with certain specified proteins. In addition, the DNA conjugated to theDNA-conjugated antibody type are unique for each type of DNA-conjugatedantibody and have a common sequences at both ends, which can be used foramplification of the DNA.

After separation of the protein-bound DNA-conjugated antibodies fromunbound DNA-conjugated antibodies, the DNA is made to release from theantibody and separated from solution. Subsequently, each DNA type can beamplified from common primers and detected by hybridization to DNA arraychips or membranes. Thus, by detecting a particular type of DNA, theimmuno-DNA array system indirectly detects the presence of thecorresponding specified proteins. In addition to assessing the specifiedproteins in solution, quantification of the amount of specified proteincan be conducted using the immuno-DNA array system by correlating theamount of each DNA type to an amount of specified protein. Methods andtechniques for immobilization and detection of agents such as proteinshas been described in Chin et al. (U.S. Pat. No. 6,197,599) andWohlstadter et al. (U.S. Pat. No. 6,140,045), both of which areincorporated herein by reference.

In other embodiments, the antibody included in the DNA-conjugatedantibody can be substituted with peptides, proteins (e.g., which canbind to specific protein), DNA (e.g., Aptamers, which can bind tospecific protein), ligands, receptors, mrRNAs, oligonucleotides,lectins, hormones, carbohydrates, lipids, small molecules, cells, drugs,and other capture reagents known in the art.

The immuno-DNA array system has several advantages over protein arrays.The immuno-DNA array system is more sensitive than protein arrays sincepooymerase chain reaction (PCR) amplification steps are included in thissystem. The immuno-DNA array system is flexible and sensitive enough todetect from microgram to attogram levels of proteins, while proteinarrays can only detect nanogram to picogram levels of a protein. Proteinarrays (e.g., prepared by spotting protein onto glass slide or othersolid support) are more difficult to prepare and store since theproteins are usually unstable. Immuno-DNA array systems/methods of thepresent invention, on the other hand, use DNA arrays (e.g., prepared byspotting DNA onto glass slides or other solid supports), which are muchmore stable than protein arrays. Furthermore, the immuno-DNA arraysystems/methods of the present invention allow detection ofprotein-protein interactions, protein-DNA interactions, andprotein-small molecular interactions, in a manner that resembles in vivoconfigurations, while current protein arrays only detect thoseinteractions in vitro. In addition, multiple rounds of experiments canbe performed separately. Subsequently, the samples from the multiplerounds can be pooled and amplified using PCR. Therefore, in the methodsof the present invention high-density arrays can be created.

As indicated above, embodiments of the immuno-DNA array system/methodsare capable of determining the presence of one or more proteins (e.g.,ten to thousands). In this regard, the immuno-DNA array system/methodscan detect multiple proteins simultaneously at microgram to alltogramlevels. Furthermore, the immuno-DNA array system/methods can detectprotein modification, such as phosphorylation, glycosylation, oxidation,ubiquitination, and acetylation. Consequently, the immuno-DNA arraysystem/method can facilitate the accurate profiling of diseasephenotypes and accelerate the identification and characterization ofprotein expression patterns, which can be used to determine cellularpathways associated with disease development. In particular, assessingproteins using the immuno-DNA array systems/methods of the presentinvention can provide a broad understanding of disease development(e.g., infection diseases, cancer, and immunological diseases). Currentdiagnostic methods can only measure the change of one protein at onetime, which greatly limits accurate diagnosis. Simultaneous detection ofmultiple antibodies, which can be correlated to specified proteins, canprovide a better analysis and greatly reduce the cost of proteinanalysis.

Embodiment A

An embodiment of the immuno-DNA array method/system is capable ofassessing the presence of one or more proteins in a solution. Theimmuno-DNA array system/method can be used in a kit or biosensor. Ingeneral, the immuno-DNA array system/method includes conjugating theproteins of interest to a solid support and then contacting the solidsupport to a DNA-conjugated antibody solution having multipleDNA-conjugated antibody types. The DNA bound to each DNA-conjugatedantibody type is unique for each DNA-conjugate antibody type. Inaddition, each type of DNA-conjugate antibody has an affinity for aparticular protein. Consequently, each DNA-conjugated antibody typebinds to a corresponding protein on the surface of the solid support.

Thereafter, the protein may be removed from the solid support and theDNA released from the DNA-conjugated antibody. The DNA is separated fromthe solution, amplified, and detected. The detection of a type of DNAindicates the presence of a particular protein. In this manner, anassessment of the proteins present in the protein solution can beconducted.

Now referring to the figures, FIGS. 1A and 1B are schematic diagramsthat illustrate a representative embodiment of the immuno-DNA assaysystem/method. FIG. 1A is a schematic that illustrates contacting (e.g.,incubating) a solution of proteins 103, 105, and 107 with a solidsupport 101. The solution of proteins can include one or more specifiedproteins 103, 105, and 107 (e.g., cytokine, EGF, insulin, MCP-1, EGFR,PDGFR, PLCγ or other solution containing proteins). In anotherembodiment, the solution of proteins can include one or more modifiedproteins (e.g., by phosphorylation, glycosylation, oxidation,ubiquitination, and acetylation) or other agents that can be used toidentify protein modification. Further, the solution of proteins caninclude DNA, RNA, lectin, hormones, antibodies, carbohydrates, lipids,other organic chemicals, small molecules, cells, and/or drugs.

Some of the proteins in the solution 103, 105, and 107 conjugate withthe solid support 101 forming a protein-conjugated solid support 109.The specified proteins 103, 105, and 107 can conjugate with the solidsupport 101 via covalent bonds and/or via non-covalent attractive forcessuch as hydrogen bond interactions, hydrophobic attractive forces, andionic forces, for example.

The solid support 101 can be any solid support 101 that has an affinityfor the proteins of interest 103, 105, and 107, and these include, forexample, magnetic beads, agarose, membranes, sepharose, glass slides,and tissue culture plates. In addition, the solid support 101 mayinclude compounds (e.g., proteins, carbohydrates, antibodies, etc.)bound to the surface of the solid support 101 that can enhance theaffinity of the proteins of interest 103, 105, and 107 to conjugate tothe solid support 101.

FIG. 1B is a schematic that illustrates contacting theprotein-conjugated solid support 109 with a solution having a pluralityof DNA-conjugated antibody types 113, 115, and 117. Each of theDNA-conjugated antibody types 113, 115, and 117 has an affinity for oneor more specified proteins 103, 105, and 107, and preferably has anaffinity for only one specified protein. In addition, the DNA bound toeach DNA-conjugate antibody type 113, 115, and 117 is unique andcorresponds with a specified protein 103, 105, and 107. Therefore,contacting the DNA-conjugated antibody solution with theprotein-conjugated solid support 109 facilitates DNA-conjugated antibodytypes 113, 115, and 117 to bind or bond with a specified protein 103,105, and 107 to form a DNA-protein-conjugated solid support 121.

The DNA-conjugated antibodies have the following features: 1) DNA andantibody have same specificity (e.g., epidermal growth factor (EGF)specific DNA conjugates to antibody against EGF or other specificsequence); 2) all DNA conjugated to antibodies contain common primers,therefore all DNA can be amplified with same pair of primers; 3) DNA canbe conjugated to the antibody through covalent bond or noncovalent bondsuch as biotin-streptavidin interaction; and 4) DNA can be referred toas an expression sequence tag (EST), synthesized oligonuleotides, mRNA,or other genomic sequence.

The antibody can include, for example, a monoclonal antibody or apolyclonal antibody. In other embodiments the antibody can besubstituted with peptides, proteins (e.g., which can bind to a specificprotein), DNA (e.g., aptamers, which can bind to specific protein),ligands, receptors, mRNAs, oligonucleotides, lectins, hormones,carbohydrates, lipids, small molecules, cells, drugs, and other capturereagents known in the art.

Thereafter, the protein is removed from the DNA-protein-conjugated solidsupport 121 and the DNA 123, 125, and 127 is released from theDNA-conjugate antibody 113, 115, and 117. The DNA 123, 125, and 127 canbe separated from solution and can be amplified and detected.

The removal of the protein from the DNA-protein-conjugated solid support121 and release of the DNA 123, 125, and 127 from the DNA-conjugateantibody types can be executed using techniques known in the art, suchas, for example, proteinase K digestion, endopeptidase digestion,aminopeptidase digestion, carboxypeptidase digestion, phenol extraction,chloroform extraction, and heat.

Each DNA type 123, 125, and 127 can be amplified using techniques knownin the art to amplify DNA such as, for example PCR. In this regard, theDNA is amplified by the use of short synthetic oligonucleotides that arecomplementary to two terminal regions of the DNA. These oligonucleotidesare extended by a thermostable DNA polymerase on the DNA template. Thiscauses new DNA chains to span the region delineated by the two chosentermini. Consequently, a 100,000-fold or better amplification of the DNAcan be achieved. In particular, each DNA type can be amplified andlabeled by PCR in the presence of a pair of primers and labeled (e.g.,cy3, cy5, 32p, 33p) using nucleotides, or other labeled nucleotidesknown in the art (e.g., biotin-labeled nucleotide). Since all DNAconjugated to antibodies contain identical sequence at both sides, thesame primers can be used to amplify all DNA fragments simultaneously.

After amplification, each amplified DNA product is hybridized to a DNAchip or DNA array. The DNA chip can be prepared from oligonucleotides orDNA clones. Similarly, the DNA array can be prepared fromoligonucleotides or DNA clones. Generally, since the identities of DNAin each spot are known, via hybridization of the DNA, the identities ofDNA from DNA-conjugated antibodies can be deduced. The hybridizationsignals can be detected using techniques known in the art for detectingDNA such as, for example, fluorescence, chemiluminescence, substratestaining, and isotope detection. In addition, the identity of DNAreleased from DNA-conjugated antibodies can also be identified by othermethods in the art such as, but not limited to, DNA sequence,electrophoresis, and techniques that use specific tags. The signals canalso be amplified by other well-known methods, such as, but not limitedto, rolling circle amplification (RCA), methods using biotinyl tyramideand hydrogen peroxide, and 3 DNA-label technology.

Each type of DNA 123, 125, and 127 corresponds to a specified protein103, 105, and 107. In this manner, each type of DNA can be related to aspecified protein indicating that the specified proteins 103, 105, and107 are present in the protein solution.

In addition, the amount of each DNA 123, 125, and 127 can be quantified,which in turn can be related to the quantity of each of the specifiedproteins 103, 105, and 107 present in the solution of proteins.

Similarly, cells also can be conjugated to a solid support. Aftercontacting with DNA-conjugated antibodies, the specific proteins presentin the cell surface can be identified as described above.

Furthermore, specific protein modification can also be detected by thisembodiment. In this regard, antibodies against specific modification,such as tyrosine phosphorylation, can be conjugated to a solid support.Tyrosine-phosphorylated proteins, for example, are separated from theunphosphorylated proteins after binding to the solid support conjugatedwith antibody against tyrosine-phosphorylation. The specific modifiedproteins are detected by contacting a solution containing a plurality ofDNA-conjugated antibody types described above.

Embodiment B

FIGS. 2A and 2B are schematic diagrams that illustrate anotherrepresentative embodiment of the immuno-DNA assay method/system. Theimmuno-DNA array system can be used in a kit or biosensor. Thisembodiment includes multiple solid substrates 201 for the specifiedproteins 203, 205, and 207 to conjugate with, rather than one solidsubstrate as shown in FIGS. 1A-1B. FIG. 2A is a schematic thatillustrates contacting multiple solid supports 201 with a solution ofproteins that includes specified proteins 203, 205, and 207. Specifiedproteins 203, 205, and 207 conjugate with the solid supports 201 formingmultiple protein-conjugated solid supports 209, 210, and 211. The solidsupport 201 can be any solid support that has an affinity for thespecified proteins of interest and these include, for example, magneticbeads, agarose, membranes, and sepharose. The solution of proteins issimilar to the solution described in relation to FIGS. 1A and 1B.

FIG. 2B is a schematic that illustrates contacting theprotein-conjugated solid supports 209, 210, and 211 with a solutionhaving a plurality of DNA-conjugated antibody types 213, 215 and 217.Each type of the DNA-conjugated antibody 213, 215, and 217 has anaffinity for one or more specified proteins 203, 205 and 207, andpreferably for only one specified protein. Therefore, contacting theDNA-conjugated antibody solution with the protein-conjugated solidsupports 209, 210, and 211 facilitates the binding or bonding of eachtype of DNA-conjugated antibody 213, 215, and 217 with the specifiedproteins to form plural complexes containing DNA-conjugated antibody andspecific protein-bound solid supports 219, 220, and 221. UnboundDNA-conjugated antibodies and the excess amount of DNA-conjugatedantibodies are separated from the DNA-conjugated antibodies and specificprotein complexes. Subsequently, the protein is removed from theDNA-protein-conjugated solid supports 219, 220, and 221 and the DNA 223,225, and 227 is released from the DNA-conjugated antibody. The DNA 223,225, and 227 is separated from the solution and can be amplified anddetected in a manner consistent with the techniques discussed inreference to FIGS. 1A and 1B.

Embodiment C

In addition to identifying and quantifying specified proteins in asolution, the embodiments of the immuno-DNA array method/system canassess protein-protein interaction. The immuno-DNA array system can beused in a kit or biosensor. In this embodiment the immuno-DNA arraysystem/method includes conjugating a first protein to a solid supportforming protein-conjugated solid supports. Then the protein-conjugatedsolid support, is contacted with a protein solution having specifiedproteins. The specified proteins bind to the first protein formingprotein-protein-conjugated solid supports. Theprotein-protein-conjugated solid supports are contacted with aDNA-conjugated antibody solution having multiple DNA-conjugated antibodytypes. The DNA bound to each DNA-conjugated antibody type is unique tothat DNA-conjugate antibody type. Each type of DNA-conjugated antibodyhas an affinity for a particular specified protein. Consequently, eachDNA-conjugated antibody type binds to a corresponding specified protein,which forms a complex on the surface of the protein-protein-conjugatedsolid support (DNA-protein-protein-conjugated solid supports).Thereafter, the protein is removed from theDNA-protein-protein-conjugated solid support and the DNA is releasedfrom the DNA-conjugated antibody. The DNA is separated from the solutionand amplified and detected. Each type of DNA detected corresponds to aparticular protein, so that detection of a type of DNA indicates thatthe specified protein interacted with the first protein. In this manner,an assessment of protein-protein interaction can be conducted.

FIGS. 3A-3C are schematic diagrams that illustrate a representativeembodiment where the immuno-DNA assay system/method can be used toassess protein-protein interactions. FIG. 3A is a schematic thatillustrates contacting multiple solid supports 301 with a first protein303 forming protein-conjugated solid supports 305.

FIG. 3B is a schematic that illustrates contacting theprotein-conjugated solid supports 305 with a solution of proteins. Thesolution of proteins includes one or more specified proteins 307, 308,309, 310, and 311. In another embodiment, the solution of proteins caninclude one or more small molecules. Contacting the protein-conjugatedsolid supports 301 with the solution of proteins facilitates theassessment of the interaction between the first protein 303 and thespecified proteins 307, 308, 309, 310, and 311 present in solution.Subsequently, multiple protein-protein-conjugated solid supports 317,318, and 319 are created with non-conjugated proteins 311 and 310remaining in solution, which can be washed away.

FIG. 3C is a schematic that illustrates contacting the protein-proteinsolid supports 317, 318, and 319 with multiple DNA-conjugated antibodytypes 327, 328, and 329. Each of the DNA-conjugated antibody types 327,328, and 329 has an affinity for one or more specified proteins 307,308, 309, 310, and 311, and preferably only one specified protein.Contacting the DNA-conjugated antibody solution with the protein-proteinsolid supports 317, 318, and 319 facilitates DNA-conjugated antibodytypes 327, 328, and 329 to bond or bind with a specified protein to formDNA-protein-protein conjugated solid supports 337, 338, and 339.Thereafter, the first protein is removed from theDNA-protein-protein-conjugated solid supports 337, 338, and 339 and theDNA 347, 348, and 349 are released from the DNA-conjugated antibody. TheDNA 347, 348, and 349 are separated from solution and can be amplifiedand detected in a manner consistent with the techniques discussed inreference to FIGS. 1A and 1B.

The same approach also can be used to detect and screen drug targets(small molecules) and protein interaction. In this embodiment, the drugcan be conjugated to a solid support directly or indirectly throughanother molecule, such as albumin. The drug conjugated to the solidsupport then can be contacted with a solution having proteins or othermolecules that may bind to the drug, such as lysate containing multipleproteins.

The detection of specific proteins interacting with the drug can beperformed by the techniques discussed above.

Embodiment D

Another embodiment of the immuno-DNA array method/system is capable ofassessing DNA-protein interactions. The immuno-DNA array system/methodcan be used in a kit or biosensor. In this embodiment the immuno-DNAarray system/method includes conjugating a first DNA (e.g., usually apromoter from a particular gene) to a solid support forming aDNA-conjugated solid support. Then the DNA-conjugated solid support iscontacted with a protein solution having specified proteins. Thespecified proteins conjugate with the first DNA, formingprotein-DNA-conjugated solid supports. The protein-DNA-conjugated solidsupports are contacted with a DNA-conjugated antibody solution havingmultiple DNA-conjugated antibody types. The DNA (i.e., the second DNA)bound to each DNA-conjugated antibody type is unique to thatDNA-conjugate antibody type. Each type of DNA-conjugated antibody typeshas an affinity for a particular specified protein. Consequently, eachDNA conjugates with a corresponding specified protein on the surface ofthe protein-DNA-conjugated solid support to formDNA-protein-DNA-conjugated solid supports. Thereafter, the complexes areseparated from the unbound molecules and the excess DNA-conjugatedantibodies. Second DNA are released from the DNA-conjugated antibodiesand separated from the solution and amplified and detected. Since thefirst DNA does not contain common primers, it cannot be amplified underthe same condition. Each type of second DNA detected corresponds to aspecified protein, so that detection of a type of second DNA indicatesthat the specified protein interacted with the first DNA. In thismanner, an assessment of the DNA-protein interaction can be conducted.

FIGS. 4A-4C are schematic diagrams that illustrate a representativeembodiment of the immuno-DNA assay system that can be used to assessDNA-protein interactions. FIG. 4A is a schematic that illustrates theconjugation of a solid support 401 with the first DNA or DNA fragments403 forming DNA-conjugated solid supports 405. In another embodiment,the first DNA 403 can be a polynucleotide as described above.

FIG. 4B is a schematic that illustrates contacting DNA-conjugated solidsupports 405 with a solution of proteins. The solution of proteinsincludes multiple specified proteins 407, 408, 409, 410, and 411.Contacting the DNA-conjugated solid supports 405 with the solution ofproteins facilitates the assessment of the interaction between the firstDNA 403 and the multiple specified proteins 407, 408, 409, 410, and 411in solution. Thereafter, multiple protein-DNA-conjugated solid supports413 are created, where specified proteins 407, 408, and 409 can bind toparticular areas of the DNA 403.

FIG. 4C is a schematic that illustrates contacting the protein-DNA solidsupports 413 with multiple DNA-conjugated antibody types 417, 418, and419. Each type of the DNA-conjugated antibody 417, 418, and 419 has anaffinity for one or more specified proteins 407, 408, 409, 410, and 411,preferably only one specified protein. Contacting the DNA-conjugatedantibody solution with the protein-DNA-conjugated solid supports 413facilitates the bonding or binding of the DNA-conjugated antibody types417, 418, and 419 with a specified protein 407, 408, 409, 410, and 411to form a DNA-protein-DNA-conjugated solid support 421. Thereafter, thecomplexes are separated from the unbound proteins and excessDNA-conjugated antibody by precipitation, extensive wash, or magneticforce. DNA 427, 428, and 429 associated with the DNA-conjugated antibodyare released and separated from solution and can be amplified anddetected in a manner consistent with the techniques discussed inreference to FIG. 1A and 1B.

Embodiment E

Another embodiment of the immuno-DNA array method/system is capable ofassessing one or more protein inhibitors. The immuno-DNA arraysystem/method can be used in a kit or biosensor. In this regard, theimmuno-DNA array system/method is capable of assessing if one or moreinhibitors can inhibit known protein-protein interactions. In thisembodiment the immuno-DNA array system/method includes conjugating afirst protein to a solid support, forming protein-conjugated solidsupports. The protein-conjugated solid supports are then contacted witha protein solution having specified proteins and inhibitor(s). The firstprotein and the specified proteins are known to interact under knownincubation conditions. By adding the inhibitor, the immuno-DNA arraysystem/method can assess whether the inhibitor(s) inhibit theinteraction of one or more of the specified proteins with the firstprotein.

The specified protein and inhibitor are allowed to conjugate with thefirst protein-forming protein-protein-conjugated andinhibitor-protein-conjugated solid supports. The solid supports arecontacted with a DNA-conjugated antibody solution having multipleDNA-conjugated antibody types. The DNA bound to each DNA-conjugatedantibody type is unique to that DNA-conjugate antibody type. Each typeof DNA-conjugated antibody has an affinity for a particular specifiedprotein. Consequently, each DNA-conjugated antibody type binds with acorresponding protein on the surface of the protein-protein-conjugatedsolid support, forming DNA-protein-protein-conjugated solid supports.

Thereafter, the complexes are separated from first protein and the DNAis released from the DNA-conjugated antibody. The DNA is separated fromthe solution and amplified and detected. Each type of DNA detectedcorresponds to a particular protein, so that detection of a type of DNAindicates that the inhibitor proteins did not inhibit theprotein-protein interaction. In this manner, an assessment of whetherinhibitors inhibit particular protein-protein interactions can beconducted.

FIGS. 5A and 5B are schematic diagrams that illustrate a representativeembodiment of the immuno-DNA assay system/method that can be used toassess the ability of an inhibitor to inhibit protein-proteininteractions. After a protein-protein interaction is known for a pair ofproteins (e.g., a first protein and a second protein), an assessment canbe made to determine inhibitors that inhibit the interaction between thepair of ther first and second proteins.

FIG. 5A is a schematic that illustrates contacting multiple solidsupports 501 with a solution of first proteins 503 formingprotein-conjugated solid supports 505.

FIG. 5B is a schematic that illustrates contacting the firstprotein-conjugated solid supports 505 with a solution of proteins. Thesolution of proteins includes multiple target proteins 507, 508, and 509and an inhibitor 510. The target proteins 507, 508, and 509 and thefirst protein 503 exhibit interaction under known incubation conditions.The inhibitor usually is a small molecule that is being assessed todetermine whether or not it inhibits the interaction between the targetproteins 507, 508, and 509 and the first protein 503. In this regard,contacting the first protein-conjugated solid supports 505 with thesolution of target proteins 507, 508, and 509 and inhibitor 510facilitates the formation of protein-protein-conjugated solid supports517 and 519 and inhibitor-protein-conjugated solid supports 518. Theremaining solution can be washed away at this time.

FIG. 5C is a schematic that illustrates contacting theprotein-protein-conjugated solid supports 517 and 519 andinhibitor-protein-conjugated solid supports 518 with one or moreDNA-conjugated antibody types 527, 528, and 529. Each of theDNA-conjugated antibody types 527, 528, and 529 has an affinity for oneor more target proteins 507, 508, and 509, and preferably for only onetarget protein. Therefore, contacting the DNA-conjugated antibody types527, 528, and 529 with the protein-protein-conjugated solid supports517, 518, and 519 facilitates bonding or binding of the DNA-conjugatedantibodies 527, 528, and 529 with a target protein 507, 508, and 509 toform DNA-protein-protein-conjugated solid supports 537 and 539. Theinhibitor 510 has inhibited the target protein 508 from interacting withthe first protein 503. Consequently, DNA-conjugated antibody 528 cannotconjugate to protein-protein-conjugated solid support 518. Thereafter,the target proteins are removed from the DNA-protein-conjugated solidsupports 537 and 539 and the DNA 547 and 549 are released from theDNA-conjugated antibodies. The DNA 547 and 549 is separated from thesolution and can be amplified and detected in a manner consistent withthe techniques discussed in reference to FIGS. 1A and 1B.

As discussed above, each detected DNA corresponds to a particular targetprotein. From these results it can be determined whether one or more ofthe target proteins are inhibited from interacting with the firstprotein. Therefore, these results can then be compared to standard orknown results for protein-protein interactions to determine if one ormore of the target proteins were inhibited by the inhibitor protein.

Embodiment F

Another embodiment of the immuno-DNA array method/system is capable ofassessing inhibitors to inhibit DNA. The immuno-DNA array system/methodcan be used in a kit or biosensor. In this regard, the immuno-DNA arraysystem/method is capable of assessing if one or more inhibitors caninhibit known DNA-protein interactions. In this embodiment theimmuno-DNA array system/method includes conjugating a DNA to a solidsupport forming DNA-conjugated solid supports. Then the DNA-conjugatedsolid support is contacted with a protein solution having targetproteins and an inhibitor. The first DNA and the target proteins areknown to interact under known incubation conditions. By adding theinhibitor, the immuno-DNA array system/method can assess whether theinhibitor inhibits the interaction of one or more of the target proteinswith the first DNA.

The target proteins and inhibitor are allowed to conjugate with thefirst DNA forming protein-DNA-conjugated and inhibitorprotein-DNA-conjugated solid supports. The solid supports are contactedwith a DNA-conjugated antibody solution having multiple DNA-conjugatedantibody types. The DNA bound to each DNA-conjugated antibody type isunique to that DNA-conjugate antibody type. Each type of DNA-conjugatedantibody has an affinity for a particular target protein. Consequently,each DNA-conjugated antibody type conjugates with a correspondingprotein on the surface of the protein-DNA-conjugated solid support,forming DNA-protein-DNA-conjugated solid supports.

Thereafter, after separating complexes from unbound molecules andDNA-conjugated antibodies, DNA associated with the DNA-conjugatedantibodies is released from the DNA-conjugated antibody and separatedfrom the solution and amplified and detected. Each type of second DNAdetected corresponds to a target protein, so that detection of a type ofsecond DNA indicates that the inhibitor proteins did not inhibit theDNA-protein interaction. The first DNA does not contain common primers,and therefore can not be amplified in the PCR step. In this manner, anassessment of whether inhibitors inhibit particular DNA-proteininteractions can be conducted.

FIGS. 6A-6C are schematic diagrams that illustrate a representativeembodiment of the immuno-DNA assay system/method and method that can beused to assess whether an inhibitor inhibits DNA-protein interactions.After a DNA-protein interaction is known for DNA and target proteins, anassessment can be made to determine if inhibitors inhibit theinteraction between DNA and one or more target proteins.

FIG. 6A is a schematic that illustrates contacting a solid support 601with the first DNA or DNA fragments 603 forming DNA-conjugated solidsupports 605. FIG. 6B is a schematic that illustrates contacting theDNA-conjugated solid support 605 with a solution of proteins. Thesolution of proteins includes multiple target proteins 607, 608, and 609and an inhibitor 610. The target proteins 607, 608, and 609 and DNA 603exhibit conjugation under known incubation conditions. The inhibitor 610is a protein that is being assessed to determine if it inhibits theconjugation between one or more target proteins 607, 608, and 609 andthe first DNA 603. In this regard, contacting the DNA-conjugated solidsupports 605 with the solution of target proteins 607, 608, and 609 andinhibitor 610 facilitates the formation of protein-DNA-conjugated solidsupport 611 that may include the inhibitor 610.

FIG. 6C is a schematic that illustrates contacting theprotein-DNA-conjugated solid supports 611 with multiple DNA-conjugatedantibody types 617, 618, and 619. Each type of the DNA-conjugatedantibody 617, 618, and 619 has an affinity for one or more targetproteins 607, 608, and 609, and preferably for only one target protein.Therefore, contacting the DNA-conjugated antibody types 617, 618, and619 with the protein-DNA-conjugated solid supports 611 facilitates thebonding or binding of the DNA-conjugated antibody types 617, 618, and619 with target protein 607, 608, and 609 to formDNA-protein-DNA-conjugated solid supports 621. The inhibitor 610 hasinhibited the target protein 609 from interacting with the DNA.Consequently, DNA-conjugated antibody 619 can not conjugate to theprotein-DNA conjugated solid support 611. Thereafter, the second DNA 627and 628 are released from the DNA-conjugated antibody and separated fromthe solution and can be amplified and detected in a manner consistentwith the techniques discussed in reference to FIGS. 1A and 1B.

As discussed above, each detected DNA corresponds to a particular targetprotein. From these results it can be determined whether one or more ofthe target proteins are inhibited from interacting with the first DNA.These results can then be compared to standard or known results forDNA-protein interactions to determine if the inhibitors inhibit one ormore of the target proteins from interacting with the DNA.

Embodiment G

An embodiment of the immuno-DNA array method/system is capable ofassessing the presence and levels of one or more autoantibodies. Theimmuno-DNA array system/method can be used in a kit or biosensor. Ingeneral, the immuno-DNA array system/method includes contacting theproteins of interest to solid supports having antibodies bound to thesurface of the solid support. To this end, protein-conjugated solidsupports are formed.

Then the protein-conjugated solid supports are contacted with aDNA-conjugated antibody solution having multiple DNA-conjugated antibodytypes to form DNA-conjugated solid supports. The DNA bound to eachDNA-conjugated antibody type is unique for each DNA-conjugate antibodytype. In addition, each type of DNA-conjugate antibody has an affinityfor a particular protein. Consequently, each DNA-conjugated antibodytype binds to a corresponding protein on the surface of the solidsupport.

For example, a patients serum, which may contain different types ofautoantibodies is contacted with a mixture of purified proteins, celllysate, or tissue lysate to form autoantibody-antigen complexes. Thecomplexes are then contacted with DNA-conjugated antibodies to formautoantibody-antigen-DNA-conjugated antibody complexes. For example,anti-human IgG conjugated to solid supports are applied to separate theautoantibody-antigen-DNA-conjugated antibody complexes from excessamounts of DNA-conjugated antibody and other unbound molecules.Thereafter, the protein is removed from the solid support and the DNA isreleased from the DNA-conjugated antibody. The DNA is separated from thesolution, amplified, and detected. The detection of a type of DNAindicates the presence of a particular protein. In this manner, anassessment of the interaction of the antigens and autoantibodies can beconducted.

FIGS. 7A and 7B are schematic diagrams that illustrate anotherrepresentative embodiment of the immuno-DNA assay system/method. FIG. 7Ais a schematic that illustrates contacting multiple autoantibodies 701A,701B, and 701Γ with a solution of proteins that includes specifiedproteins 703, 705, and 707. Alternatively, the autoantibodies can beattached to a substrate, as described in the examples above.

Specified proteins 703, 705, and 707 bind to correspondingautoantibodies 701A, 701B, and 701 Γ forming multiple protein(antigen)-autoantibody complexes 709, 710, and 711. The solution ofautoantibody contains one and more types of autoantibodies, 701A, 701B,and 701 Γ, where each autoantibody (e.g., A, B, and Γ) has an affinityfor corresponding proteins (antigens).

FIG. 7B is a schematic that illustrates contacting theprotein-autoantibody complexes 709, 710, and 711 with a solution havinga plurality of DNA-conjugated antibody types 713, 715 and 717. Each typeof the DNA-conjugated antibody 713, 715, and 717 has an affinity for oneor more specified proteins 703, 705 and 707, and preferably for only onespecified protein. Therefore, contacting the DNA-conjugated antibodysolution with the protein-autoantibody complexes 709, 710, and 711facilitates the binding or bonding of each type of DNA-conjugatedantibody 713, 715, and 717 with the specified proteins to form pluralcomplexes (i.e., protein-autoantibody-DNA-conjugated antibody)containing DNA-conjugated antibody, specific protein, and autoantibody,719, 720, and 721.

FIG. 7C is a schematic that illustrates contacting theprotein-autoantibody-DNA-conjugated antibody complex 719, 720, and 721with a solution of anti-species specific antibodies α, β, and γ (e.g.,if the autoantibodies are from human serum, anti-human IgG will be used)conjugated to a solid support 729, 730, and 731. The solution ofantibodies 729, 730, and 731 includes one or more types of antibodies,where each autoantibody (e.g., A, B, and Γ) has an affinity for the samecorresponding antibody (α, β, and γ) (e.g., human IgG). Thus, eachantibody 729, 730, and 731 binds to a corresponding autoantibody on thesurface of the DNA-conjugated solid supports 719, 720, and 721. Theresultant product of contacting the protein-autoantibody-DNA-conjugatedantibody complex 719, 720, and 721 with the solution of antibodies 729,730, and 731 are antibody-protein-autoantibody-DNA conjugated antibodycomplexes 739, 740, and 741.

Subsequently, the protein is removed from the complexes 739, 740, and741 and the DNA 743, 745, and 747 is released from the DNA-conjugatedantibodies. The DNA 743, 745, and 747 is separated from the solution andcan be amplified and detected in a manner consistent with the techniquesdiscussed in reference to FIGS. 1A and 1B.

The detection of each type of DNA indicates the presence of a particularprotein.

In this manner, an assessment of the presence and levels ofautoantibodies can be conducted.

Embodiment H

The following embodiment illustrates the application of the immuno-DNAmicroarray method/system to assess multiple protein phosphorylation. Theimmuno-DNA array system/method can be used in a kit or biosensor. Twowell-known model systems have been selected to test the immuno-DNAmicroarray system/method. One model includes the use of A431 cellsstimulated with EGF. Treatment of A431 cells with EGF leads to thephosphorylation of EGFR and activates the Ras signal transductionpathway.

Another model includes treatment of NIH3T3 cells with PDGF brings aboutthe phosphorylation of PDGFR and activates Ras signal transductionpathway.

Several important proteins such as EGFR, PDGFRα and PLCγ in those signalpathways can be selected for conjugation with their corresponding cDNAobtained from EST. The EST clones are available from several vendors.When multiple EST clones are available, the EST with the shortestsequences (e.g., 200 to 1,000 bp) and high specificity can be selectedsince longer sequences may affect the ability of cDNA-conjugatedantibodies to bind with their corresponding antigens. Since all of theEST can be amplified by the same primers, the use of EST clones cansimplify the PCR process during the preparation of conjugation ofantibodies to cDNA and the generation of probes for DNA microarrays.Antibodies with the highest titer and specificity can be selected forconjugation (e.g., monoclonal antibodies).

To make cDNA conjugated antibodies, the antibodies can be treated with a10-fold molar excess of sulfo-GMBS. After removing untreated sulfo-GMBSby chromatography over a PD-10 column, the antibody can then beconcentrated in a centricon. The sulfo-GMBS-activated antibody and5'thiol cDNA can be conjugated. Antibodies conjugated to cDNA can thenbe purified by anion exchange chromatography on Q-Sepharose. Using asalt gradient, the free cDNA can be further removed by size exchangechromatography on Superdex-200.

Another approach to conjugate DNA to antibodies is to use aldehydemodification oligonucleotides/DNA. Proteins are modified by SHNH(Succininmidyl hydraziniumnicotinate hydrochloride) in 100 ml DMF(N,N-Dimethylformamide). Modified proteins are conjugated toaldehyde-modified oligonucleotides/DNA by incubation over night at roomtemperature. The reactions are assayed by DNA gel analysis. Theconjugates are purified by ion-exchange DEAE Sepharose™ Fast column orQ-sephorase™ Fast Flow column. DAN-conjugated antibody fractions arecollected and are subjected to DNA gel analysis and dialysis in 1×PBS(phosphate buffer salt) buffer.

The effect of the conjugation on the ability of the antibody to bindwith the antigen can be determined by immunoprecipitation. Conjugatedantibody and unconjugated antibody can be incubated with cell lysatesprepared from growth factor (GF)-stimulated cells. Theimmunoprecipitated complexes can then be separated by sodium dodecylsulphate polyacrimide gel electrophoresis (SDS PAGE).

After transferring the proteins to PDVF membranes, the membranes can beprobed with unconjugated antibody and DNA-conjugated antibody,respectively. The intensities of signals can be compared. If theintensities of signals are similar between DNA-conjugated andunconjugated antibodies, this indicates that the conjugation of cDNAdoes not affect the ability of the antibody to bind to its antigen. Ifthe conjugation of cDNA significantly reduces the ability of theantibody to bind its antigen, the size of cDNA can be reduced. From theimmuno-PCR data, the conjugation of cDNA up to 1 to 2 kb of the antibodyshould not significantly affect the ability of the antibody to bind toits antigen. High sensitivity should be able to be obtained using thePCR amplification technique even if the attached DNA affects the bindingactivity of the antibody to the antigen.

To optimize the development of the cDNA conjugated antibodies severalsets of experiments can be performed. One includes varying theconcentration of the DNA-conjugated antibodies. Several concentrationssuch as 1 ng/ml, 10 ng/ml, 100 ng/ml, 100 μg/ml and 10 μg/ml can be usedto select the optimal concentration. Another parameter that can betested includes varying the PCR cycles. Different PCR cycles such as 10cycles, 20 cycles, and 30 cycles can be tested. Non-PCR labels can alsobe used to detect the signal and are well known in the art. Since themodel cell lines express high levels of either EGFR or PDGFR, differentdiluted cell lysates can be used to test the detection limits of thisembodiment.

To make cDNA microarray membranes, EST sequences used for constructionof DNA-conjugated antibodies can be amplified using a pair of universalprimers. The amplified products can be checked by agarose gelelectrophoresis and quantitated by OD at 260 over 280 10 picograms ofDNA can be spotted onto nitrocellulose membranes. EST sequences, whichdo not match for the construction of DNA-conjugated antibodies can beused as a negative control.

After the DNA conjugated antibodies and mini-DNA array membranes aremade, the system can be tested. The cell lysates and tumor tissuelysates can be prepared by homogenization in RIPA buffer containingappropriate proteinase and phosphatase inhibitors. Lysates can passthrough a 26 gauge needle to disperse any large aggregates. Cell lysatesfrom EGF-stimulated A431 cells and PDGF-stimulated NIH3T3 cells can beincubated with agarose-conjugated anti-phosphotyrosine antibody at about4° C. for about 1 hour. The phosphotyrosine proteins can then beimmunoprecipitated by agarose-conjugated anti-phosphotyrosine antibody.

After wash with RIPA, the immunoprecipitated complexes can be incubatedwith DNA conjugated specific antibodies at about 4° C. for about 1 hourto overnight. The immunoprecipitated complexes containingtyrosine-phosphorylated proteins, agarose-conjugated anti-phosphorylatedtyrosine antibody and DNA-conjugated specific antibodies can be pulleddown by centrifugation. The excess amounts of unbound cDNA conjugatedantibodies can be washed away.

Three control experiments can be conducted to determine theeffectiveness of the system and method of the present invention. Onecontrol experiment includes using agarose instead of agarose-conjugatedanti-phospho-tyrosine antibody. Any signals resulting in this conditioncan be attributed to the unspecific binding of DNA-conjugated antibodiesto agarose rather than their specific antigens or the unspecificcomplexes between proteins, agarose, and antibodies. Another controlexperiment uses H₂O instead of DNA-conjugated antibodies. The signalsresulting from this experiment can be attributed to the unspecificbinding of cellular DNA to the agarose-conjugated antibody. Anothercontrol experiment uses RIPA instead of cell lysate. The signalsresulted from this experiment again can be attributed the unspecificbinding between agarose-conjugated anti-phospho-tyrosine antibody andDNA-conjugated antibody.

The complexes can be heated at 85-100 C to set free (release) DNA fromconjugated antibodies. After heating, the DNA can be recovered. The DNAcan then be amplified and labeled by PCR in the presence of 33p dUTP orcy3 dUTP. The amplified products can hybridize to the DNA arraymembranes containing cDNA immobilized onto membrane (e.g.,nitrocellulose membrane). The system can be detected by exposure to kodax-film or phospho-imaging system, chemiluminescence imaging system, orlaser scanner. The recovered DNA also can hybridize the DNA chipscontaining cDNA or oligonucleotides. Since specific cDNAs are attachedto a corresponding antibody, the intensities of signal reflect thelevels of corresponding proteins. Since all of proteins areimmunoprecipitated with anti-phosphotyrosine antibody, the intensitiesof signal reflect the phosphotyrosine status of specific proteins. SinceA43 1 cells express high amount of EGFR, tyrosine phosphorylation inEGFR should be detected. NIH3T3 cells express a high amount of PDGFR,thus tyrosine phosphorylation in PDGFR can also be detected. Therefore,positive and negative signals can be detected.

One population of DNA can be labeled with cy3 (e.g., the DNA recoveredfrom experiment using A431 cells), while another population of DNA canbe labeled with cy5, (e.g., the DNA recovered from experiment usingNIH3T3 cells). Both probes can then hybridize to the same cDNA arraymembranes or DNA chip. The signal can be scanned using a laser scanneror CCD camera and the intensities of the signals can be comparedsimultaneously.

The tyrosine-phosphorylated status of proteins can be confirmed andverified by immuno-Western blotting analysis. In this type ofexperiment, cell lysates can be immunoprecipitated withanti-phospho-tyrosine antibody. Then the immunoprecipitated complexescan be separated by SDS-PAGE. After transferring the proteins ontomembranes, the membranes can be probed with specific antibodies. Theresults from immuno-DNA array systems/methods and immuno-Western blotcan be compared, which can be used to assess the specificity andsensitivity of immuno-DNA array systems/methods.

After the cell lines are tested, the methodology can be used to assesshuman tumor tissues. Breast cancer tissues can be used since most ofbreast cancer tissues express high amounts of phosphorylated EGFR. Tumortissue lysates can be incubated with agarose-conjugatedanti-phospho-tyrosine antibody. The immunoprecipitated complexes canthen be incubated with specific cDNA conjugated antibody. The recoveredcDNA can then be amplified by PCR and used as probes to hybridize to DNAarray membranes. The results can be confirmed by immuno-Western blotanalysis.

Embodiment I

The following embodiment illustrates the application of the immuno-DNAarray method/system to assess multiple protein expression levels. Theimmuno-DNA array system/method can be used in a kit or biosensor.Several antibodies and recombinant proteins can be selected to test theimmuno-DNA microarray system/method. EGF and anti-EGF antibody, insulinand anti-insulin, MCP-1 and anti-MCP-1 can be used. The EST clones areavailable from several vendors. When multiple EST clones are available,those with the shortest sequences (e.g., 200 to 1,000 bp) and highestspecificity can be selected since longer sequences may affect theability of cDNA-conjugated antibodies to bind to their correspondingantigens. Since all of the ESTs can be amplified by the same primers,the use of EST clones can simplify the PCR process during thepreparation of conjugated antibodies to cDNA and the generation ofprobes for DNA microarrays.

DNA conjugate antibodies can be generated in a manner as describedabove. To optimize the conditions of the array, several sets ofexperiments can be performed. One is the concentration of DNA-conjugatedantibodies. Several concentrations, such as 1 ng/ml, 10 ng/ml, 100 ng/mland 1000 ng/ml, will be used to select the optimal concentration.Another parameter that can be tested includes using different PCRcycles. Different cycles such as 10 cycles, 20 cycles and 30 cycles canbe tested.

To make mini-cDNA microarray chips, EST sequences used for constructionof DNA-conjugated antibodies can be amplified using a pair of universalprimers. The amplified products can be checked by agarose gelelectrophoresis and quantitated by OD at 260 over 280 50 pg of DNA canbe spotted onto Parckard Hydrogel chip or any other types of glassslides. EST sequences that do not match for the construction ofDNA-conjugated antibodies can be used as negative control.

After the DNA conjugated antibodies and mini-DNA array chips are made,the system can be tested. Purified recombinant proteins (100 nanogramsof protein) can be conjugated to magnetic beads. Bead-conjugatedproteins can then be incubated with DNA-conjugated antibodies at about4° C. for about 2 hours. Excess DNA-conjugated antibodies can be removedby magnetic field and washed with PBS. A control experiment can usemagnetic beads rather than magnetic bead conjugated proteins. Thesignals resulting from this experiment can contribute to the unspecificbinding between magnetic beads and DNA-conjugated antibodies. Thecomplexes can be digested with proteinase K to remove the proteins andrelease DNA from conjugated antibodies. After passing throughultrafree-probind column (Millipore) to remove proteins and smallpeptides, the DNA can be recovered by ethanol precipitation. The DNA canthen be amplified and labeled by PCR in the presence of cy3 dUTP. Theamplified products can hybridize to the mini-DNA array chips containingcorresponding cDNA. Then the signal can be scanned using a laserscanner.

Other immobilization approaches such as conjugation of proteins toagarose or to PDVF membranes can be tested. From this set ofexperiments, the immobilization condition that produces the highestsignals and that is easiest to perform can be selected.

The detection sensitivity of this approach can be analyzed. Differentamounts of purified recombinant protein can be used to test thedetection sensitivity. In addition, inter-chip variability andintra-chip variability can be determined.

The next stage of development can focus on assessing multipleantibodies. Approximately 1000 antibodies, which play important roles insignal transduction, cell growth control, DNA repair and apoptosis, canbe selected. Specific cDNA-conjugated antibodies can be generated.Antibodies with high titer and specificity (monoclonal antibodies) canbe selected for conjugation to DNA. The system can be analyzed usingcell lysates and tissue lysates. The immuno-DNA array system/methodshould be able to simultaneously detect at least 1000 proteins with highspecificity and sensitivity.

The differential expression of proteins between normal mammary gland andbreast cancer tissue can be examined by immuno-DNA array system/method.One hundred micrograms of total tissue lysates prepared from normalmammary gland and breast cancer tissue can be conjugated to magneticbeads, respectively. Bead-conjugated tissue lysates can then beimmunoprecipitated with a mixture of DNA-conjugated antibodies. Unboundantibodies can then be removed by magnetic field and proteins can beremoved by proteinase digestion. DNA can be recovered by precipitation.One pool of DNA (e.g., normal mammary gland) can be labeled with cy3 andanother pool of DNA (e.g., breast cancer tissue) can be labeled withcy5. The two pools can be combined and hybridized with DNA chips.Signals can be scanned and analyzed and the expression pattern can beclassified by clustering. Several differential proteins can be furtherconfirmed by Western blot analysis.

It should be emphasized that the above-described embodiments of thepresent invention, particularly, any “preferred” embodiments, are merelypossible examples of implementations, merely set forth for a clearunderstanding of the principles of the invention. Many variations andmodifications may be made to the above-described embodiment(s) of theinvention without departing substantially from the spirit and principlesof the invention. All such modifications and variations are intended tobe included herein within the scope of this disclosure and the presentinvention and protected by the following claims.

1. A method of detecting proteins, comprising: exposing a solid supportto a solution containing proteins; conjugating proteins to the solidsupport; exposing the solid support to a plurality of types ofDNA-conjugated antibodies, wherein each type of DNA-conjugatedantibodies has an affinity for a specified protein; forming a complexbetween a protein conjugated with the solid support and a type ofDNA-conjugated antibody when the protein is the specified protein forwhich the DNA-conjugated antibody has an affinity; separating thecomplex from the solution of proteins and the DNA-conjugated antibodies;releasing DNA from the DNA-conjugated antibodies; and detecting the DNA,wherein each DNA indicates the presence of the specified protein.
 2. Themethod of claim 1, wherein the proteins are modified-proteins.
 3. Themethod of claim 1, wherein detecting the DNA further comprises:amplifying the DNA using techniques selected from polymerase chainreaction (PCR) techniques, rolling circle amplification (RCA),techniques using biotinyl tyramide and hydrogen peroxide, and 3DNA-label techniques.
 4. The method of claim 1, wherein detecting theDNA further comprises: detecting the DNA using techniques selected fromfluorescence, chemiluminescence, substrate staining, isotope detection,surface plasmon resonance, resonance light scattering, electronicsensor, magnetic sensor, and microcantilevering.
 5. The method of claim1, wherein the solid support is selected from magnetic beads, agarose,membranes, sepharose, glass slides, and tissue culture plates.
 6. Themethod of claim 1, wherein releasing DNA comprises using techniquesselected from protease digestion, proteinase digestion, phenolextraction, chloroform extraction, and heat.
 7. The method of claim 1,wherein the antibodies in the plurality of DNA-conjugated antibodies canbe substituted with compounds selected from DNA, RNA, lectins, hormones,carbohydrates, lipids, small molecules, cells, drugs, and other capturereagents.
 8. A method of detecting protein-protein interactions,comprising: exposing a solid support to a first solution having aplurality of first proteins, wherein the plurality of first proteinsconjugate with the solid support; exposing the solid support to a secondsolution of proteins, wherein the proteins in the second solution maybecapable of conjugating with the plurality of first proteins; exposingthe solid support to a plurality of types of DNA-conjugated antibodies,wherein each type of DNA-conjugated antibody has an affinity for aspecified protein; forming a complex between a protein of the secondsolution that has conjugated with the first protein and a type ofDNA-conjugated antibody when the protein of the second solution is thespecified protein for which the DNA-conjugated antibody has an affinity;separating the complex from the solution of first proteins, secondproteins, and the DNA-conjugated antibodies; releasing DNA from theDNA-conjugated antibodies; and detecting the DNA, wherein each DNAdetected indicates the presence of the specified protein, whichindicates that the specified proteins interacted with the first protein.9. The method of claim 8, wherein the solution of proteins containsinhibitor compounds that inhibit the interaction of specified proteinswith the first protein.
 10. The method of claim 8, wherein the proteinsare modified proteins.
 11. The method of claim 8, wherein detecting theDNA further comprises: amplifying the DNA using techniques selected frompolymerase chain reaction (PCR) techniques, rolling circle amplification(RCA), techniques using biotinyl tyramide and hydrogen peroxide, and 3DNA-label techniques.
 12. The method of claim 8, wherein detecting theDNA further comprises: detecting the DNA using techniques selected fromfluorescence, chemiluminescence, substrate staining, isotope detection,surface plasmon resonance, resonance light scattering, electronicsensor, magnetic sensor, and microcantilevering.
 13. The method of claim8, wherein the solid support is selected from magnetic beads, agarose,membranes, sepharose, glass slides, and tissue culture plates.
 14. Themethod of claim 8, wherein releasing DNA comprises using techniquesselected from protease digestion, proteinase digestion, phenolextraction, chloroform extraction, and heat.
 15. The method of claim 8,wherein the antibodies in the plurality of DNA-conjugated antibodies canbe substituted with compounds selected from DNA, RNA, lectins, hormones,carbohydrates, lipids, small molecules, cells, drugs, and other capturereagents.
 16. A method of detecting protein-DNA interactions,comprising: exposing a solid support to a first DNA having at least oneportion, wherein the first DNA conjugates with the solid support;exposing the solid support to a solution of proteins, wherein theproteins in the solution are capable of conjugating with a portion ofthe first DNA; exposing the solid support to a plurality of types ofDNA-conjugated antibodies, wherein each type of DNA-conjugated antibodyhas an affinity for a specified protein; forming a complex between aprotein in the solution that has conjugated with a portion of the firstDNA and a type of DNA-conjugated antibody when the protein is thespecified protein for which the DNA-conjugated antibody has an affinity;separating the complex from the solution of proteins and theDNA-conjugated antibodies; releasing DNA from the DNA-conjugatedantibodies; and detecting the DNA, wherein each DNA detected indicatesthe presence of the specified proteins, which indicates that thespecified proteins interacted with the portion of DNA.
 17. The method ofclaim 16, wherein the solution of proteins contains inhibitor compoundsthat are capable of inhibiting the interaction of a specified proteinwith the first DNA.
 18. The method of claim 16, wherein the proteins aremodified proteins.
 19. The method of claim 16, wherein detecting the DNAfurther comprises: amplifying the DNA using techniques selected frompolymerase chain reaction (PCR) techniques, rolling circle amplification(RCA), techniques using biotinyl tyramide and hydrogen peroxide, and 3DNA-label techniques.
 20. The method of claim 16, wherein detecting theDNA further comprises: detecting the DNA using techniques selected fromfluorescence, chemiluminescence, substrate staining, isotope detection,surface plasmon resonance, resonance light scattering, electronicsensor, magnetic sensor, and microcantilevering.
 21. The method of claim16, wherein the solid support is selected from magnetic beads, agarose,membranes, sepharose, glass slides, and tissue culture plates.
 22. Themethod of claim 16, wherein releasing DNA comprises using techniquesselected from protease digestion, proteinase digestion, phenolextraction, chloroform extraction, and heat.
 23. The method of claim 16,wherein the antibodies in the plurality of DNA-conjugated antibodies canbe substituted with compounds selected from DNA, RNA, lectins, hormones,carbohydrates, lipids, small molecules, cells, drugs, and other capturereagents.
 24. The method of claim 16, wherein the first DNA can be apolypeptide.
 25. A method of detecting modified-proteins, comprising:exposing a solid support to a solution containing modified-proteins;conjugating modified-proteins to the solid support; exposing the solidsupport to a plurality of types of DNA-conjugated antibodies, whereineach type of DNA-conjugated antibody has an affinity for a specifiedmodified-protein; forming a complex between a modified-proteinconjugated to the solid support and a type of DNA-conjugated antibodywhen the modified-protein is the specified modified-protein for whichthe DNA-conjugated antibody has an affinity; separating the complex fromthe solution of modified-proteins and the DNA-conjugated antibodies;releasing DNA from the DNA-conjugated antibodies; and detecting the DNA,wherein each DNA detected indicates the presence of the specifiedmodified-proteins.
 26. The method of claim 25, wherein themodified-protein is modified by a modification process selected fromphosphorylation, glycosylation, oxidation, ubiquitination, andacetylation.
 27. The method of claim 25, wherein detecting the DNAfurther comprises: amplifying the DNA using techniques selected frompolymerase chain reaction (PCR) techniques, rolling circle amplification(RCA), techniques using biotinyl tyramide and hydrogen peroxide, and 3DNA-label techniques.
 28. The method of claim 25, wherein detecting theDNA further comprises: detecting the DNA using techniques selected fromfluorescence, chemiluminescence, substrate staining, isotope detection,surface plasmon resonance, resonance light scattering, electronicsensor, magnetic sensor, and microcantilevering.
 29. The method of claim25, wherein the solid support is selected from magnetic beads, agarose,membranes, sepharose, glass slides, and tissue culture plates.
 30. Themethod of claim 25, wherein removing DNA comprises using techniquesselected from protease digestion, proteinase digestion, phenolextraction, chloroform extraction, and heat.
 31. The method of claim 25,wherein the antibodies in the plurality of DNA-conjugated antibodies canbe substituted with compounds selected from DNA, RNA, lectins, hormones,carbohydrates, lipids, small molecules, cells, drugs, and other capturereagents.
 32. A method of detecting autoantibodies, comprising: exposinga solution containing proteins to a solution containing a plurality ofautoantibodies, wherein each autoantibody has an affinity for aspecified protein; forming a first complex between a protein in thesolution and a type of autoantibody when the protein is the specifiedprotein for which the autoantibody has an affinity; exposing the firstcomplex to a solution containing a plurality of types of DNA-conjugatedantibodies, wherein each type of DNA-conjugated antibody has an affinityfor a specified protein; forming a second complex between first complexand a type of DNA-conjugated antibody when the protein is the specifiedprotein for which the DNA-conjugated antibody has an affinity; exposingthe second complex to a solution having a plurality of antibodies,wherein each type of antibody has an affinity for a specifiedautoantibody; forming a third complex between the second complex and atype of antibody when the autoantibody is the specified antibody forwhich the antibody has an affinity; separating the third complex fromthe solution; releasing DNA from the DNA-conjugated antibodies; anddetecting the DNA, wherein each DNA detected indicates the presence of aspecified autoantibody.
 33. The method of claim 32, wherein detectingthe DNA further comprises: amplifying the DNA using techniques selectedfrom polymerase chain reaction (PCR) techniques, rolling circleamplification (RCA), techniques using biotinyl tyramide and hydrogenperoxide, and 3 DNA-label techniques.
 34. The method of claim 32,wherein detecting the DNA further comprises: detecting the DNA usingtechniques selected from fluorescence, chemiluminescence, substratestaining, isotope detection, surface plasmon resonance, resonance lightscattering, electronic sensor, magnetic sensor, and microcantilevering.35. The method of claim 32, wherein releasing DNA further is executedusing techniques selected from protease digestion, proteinase digestion,phenol extraction, chloroform extraction, and heat.
 36. A method ofdetecting protein-small molecule interactions, comprising: exposing asupport to a plurality small molecules, wherein the plurality of smallmolecules conjugate with the support; exposing the support to a solutionof proteins, wherein each of the proteins in the second solution arecapable of conjugating with a specified small molecule; forming a firstcomplex between the a small molecule and a type of protein when thesmall molecule is the specified small molecule for which the protein hasan affinity; exposing the support to a plurality of types ofDNA-conjugated antibodies, wherein each type of DNA-conjugatedantibodies has an affinity for a specified protein; forming a secondcomplex between a protein conjugated to the support and a type ofDNA-conjugated antibody when the protein is the specified protein forwhich the antibody has an affinity; separating the second complex fromthe solution of proteins and the DNA-14 conjugated antibodies; removingDNA from the DNA-conjugated antibodies; and detecting the DNA, whereineach DNA detected indicates the presence of the specified proteins,which indicates that the specified proteins interacted with the smallmolecules.
 37. The method of claim 36, wherein detecting the DNA furthercomprises: amplifying the DNA using techniques selected from polymerasechain reaction (PCR) techniques, rolling circle amplification (RCA),techniques using biotinyl tyramide and hydrogen peroxide, and 3DNA-label techniques.
 38. The method of claim 36, wherein detecting theDNA further comprises: detecting the DNA using techniques selected fromfluorescence, chemiluminescence, substrate staining, isotope detection,surface plasmon resonance, resonance light scattering, electronicsensor, magnetic sensor, and microcantilevering.
 39. The method of claim36, wherein releasing DNA comprises using techniques selected fromprotease digestion, proteinase digestion, phenol extraction, chloroformextraction, and heat.
 40. A kit for use in a method according to claim1, the kit comprising the reageants and instructions for the performanceof the method and interpretation of the results.
 41. A kit for use in amethod according to claim 16, the kit comprising the reageants andinstructions for the performance of the method and interpretation of theresults.
 42. A kit for use in a method according to claim 25, the kitcomprising the reageants and instructions for the performance of themethod and interpretation of the results.
 43. A kit for use in a methodaccording to claim 32, the kit comprising the reageants and instructionsfor the performance of the method and interpretation of the results. 44.A kit for use in a method according to claim 36, the kit comprising thereageants and instructions for the performance of the method andinterpretation of the results.
 46. An assay for detecting proteins,comprising the method claim
 1. 47. An assay for detectingprotein-protein interactions, comprising the method claim
 8. 48. Anassay for detecting protein-DNA interactions, comprising the methodclaim
 16. 49. An assay for detecting modified-proteins, comprising themethod claim
 25. 50. An assay for detecting autoantibodies, comprisingthe method claim
 32. 51. An assay for detecting protein-small moleculeinteractions, comprising the method claim 36.